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Originally published In Press as doi:10.1074/jbc.M106743200 on August 28, 2001

J. Biol. Chem., Vol. 276, Issue 44, 40847-40857, November 2, 2001
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Toxicity of Antiviral Nucleoside Analogs and the Human Mitochondrial DNA Polymerase*

Allison A. JohnsonDagger , Adrian S. Ray§, Jeremiah HanesDagger , Zucai Suo, Joseph M. Colacino, Karen S. Anderson§, and Kenneth A. JohnsonDagger ||

From the Dagger  Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, § Yale University School of Medicine Department of Pharmacology, New Haven, Connecticut 06520, and  Infectious Diseases Research, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285

To examine the role of the mitochondrial polymerase (Pol gamma ) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma  for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-beta -D-arabinofuranosyl)-5-iodouracil (FIAU), beta -L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol gamma  holoenzyme in single turnover kinetic studies to measure Kd (Km) and kpol (kcat) to estimate the specificity constant (kcat/Km) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2',3'-didehydro-2',3'-dideoxythymidine (d4T) (+)3TC (-)3TC > PMPA > azidothymidine (AZT) Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s-1 for the series FIAU > (+)3TC ~ (-)3TC > CBV > AZT > PMPA ~ d4T ddA (ddI) ddC. Removal of ddC was too slow to measure (<0.00002 s-1). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s-1) faster than it is removed (0.06 s-1) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.

* This work was supported by National Institutes of Health Grants GM44613 (to K. A. J.) and GM49551 (to K. S. A.) and by Welch Foundation Grant F-1432 (to K. A. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Institute for Cellular and Molecular Biology MBB 3.122, A4800 University of Texas, Austin, TX 78712. Tel.: 512-471-0434; Fax: 512-471-0435; E-mail: kajohnson@mail.utexas.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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